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Bioengineering human intestinal mucosal grafts using patient-derived organoids, fibroblasts and scaffolds

Nature Protocols volume 18, pages 108–135 (2023)Cite this article

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Abstract

Tissue engineering is an interdisciplinary field that combines stem cells and matrices to form functional constructs that can be used to repair damaged tissues or regenerate whole organs. Tissue stem cells can be expanded and functionally differentiated to form ‘mini-organs’ resembling native tissue architecture and function. The choice of the scaffold is also pivotal to successful tissue reconstruction. Scaffolds may be broadly classified into synthetic or biological depending upon the purpose of the engineered organ. Bioengineered intestinal grafts represent a potential source of transplantable tissue for patients with intestinal failure, a condition resulting from extensive anatomical and functional loss of small intestine and therefore digestive and absorptive capacity. Prior strategies in intestinal bioengineering have predominantly used either murine or pluripotent cells and synthetic or decellularized rodent scaffolds, thus limiting their translation. Microscale models of human intestinal epithelium on shaped hydrogels and synthetic scaffolds are more physiological, but their regenerative potential is limited by scale. Here we present a protocol for bioengineering human intestinal grafts using patient-derived materials in a bioreactor culture system. This includes the isolation, expansion and biobanking of patient-derived intestinal organoids and fibroblasts, the generation of decellularized human intestinal scaffolds from native human tissue and providing a system for recellularization to form transplantable grafts. The duration of this protocol is 12 weeks, and it can be completed by scientists with prior experience of organoid culture. The resulting engineered mucosal grafts comprise physiological intestinal epithelium, matrix and surrounding niche, offering a valuable tool for both regenerative medicine and the study of human gastrointestinal diseases.

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Fig. 1: Overview of the protocol steps.
Fig. 2: Derivation and expansion of patient-derived intestinal organoids and fibroblasts.
Fig. 3: Generation of decellularized human intestinal scaffolds.
Fig. 4: Recellularization of the scaffold in static culture.
Fig. 5: Bioreactor setup for dynamic culture.
Fig. 6: Characterization of the human intestinal mucosal grafts.

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Data availability

The data presented in Anticipated results section were previously published and are available via the original primary publication29 and its supplementary information files.

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Acknowledgements

We thank J. Brock, from the Research Illustration and Graphics team, the Francis Crick Institute, for contributions to figure illustration and M. Orford for assistance in developing the citrulline assay. We also thank the Francis Crick Institute’s Experimental Histopathology Science Technology Platform for technical support. Selected illustrations in Figs. 1 and 2 were created with BioRender.com. This work was funded by Horizon 2020 grant INTENS 668294 on the project ‘Intestinal Tissue Engineering Solution for Children with Short Bowel Syndrome.’ The laboratory of V.S.W.L. is supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001105), the UK Medical Research Council (FC001105) and the Wellcome Trust (FC001105). P.D.C. is supported by an NIHR Professorship, NIHR UCL BRC-GOSH, the Great Ormond Street Hospital Children’s Charity and the Oak Foundation. L.M. was funded by NIHR UCL BRC-GOSH Crick Clinical Research Training Fellowship and is currently funded by an NIHR Academic Clinical Lectureship. L.T. is funded by NIHR UCL BRC-GOSH Crick Clinical Research Training Fellowship and Horizon 2020 grant INTENS 668294. We are grateful to Gastroenterology, the SNAPS Unit and patients at Great Ormond Street Hospital Children NHS Trust for the intestinal samples and to P. Shi Chia for help with research consents and sample collection.

Author information

Author notes

  1. Laween Meran

    Present address: Medical Research Council Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK

  2. These authors contributed equally: Laween Meran, Lucinda Tullie.

Authors and Affiliations

  1. Stem Cell and Cancer Biology Laboratory, The Francis Crick Institute, London, UK

    Laween Meran, Lucinda Tullie & Vivian S. W. Li

  2. Stem Cell and Regenerative Medicine Section, DBC, Great Ormond Street Institute of Child Health, University College London, London, UK

    Laween Meran, Lucinda Tullie, Simon Eaton & Paolo De Coppi

  3. Specialist Neonatal and Paediatric Surgery Unit, Great Ormond Street Hospital, London, UK

    Paolo De Coppi

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  1. Laween Meran
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Contributions

L.M. developed the protocols. L.M. and L.T. performed the experiments and wrote the manuscript. S.E. advised on citrulline assay analyses. P.D.C. and V.S.W.L. edited the manuscript, supervised the study and acquired funding for the study.

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Correspondence to Paolo De Coppi or Vivian S. W. Li.

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Nature Protocols thanks Shinya Sugimot and Shiro Yui for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Meran, L. et al. Nat. Med. 26, 1593–1601 (2020): https://doi.org/10.1038/s41591-020-1024-z

Totonelli, G. et al. Biomaterials 33, 3401–3410 (2012): https://doi.org/10.1016/j.biomaterials.2012.01.012

Giobbe, G. G. et al. Nat. Commun. 10, 5658 (2019): https://doi.org/10.1038/s41467-019-13605-4

Supplementary information

Supplementary Information

Supplementary Methods and Table 1

Reporting Summary

Supplementary Video 1

Bioreactor circuit components setup 1

Supplementary Video 2

Bioreactor circuit components setup 2

Supplementary Video 3

Bioreactor circuit components setup 3

Supplementary Video 4

Bioreactor circuit components setup 4

Supplementary Video 5

Preparation of scaffold

Supplementary Video 6

Mounting of scaffold

Supplementary Video 7

Injection of fibroblasts into scaffold

Supplementary Video 8

Dynamic culture circuit setup 1

Supplementary Video 9

Dynamic culture circuit setup 2

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Meran, L., Tullie, L., Eaton, S. et al. Bioengineering human intestinal mucosal grafts using patient-derived organoids, fibroblasts and scaffolds. Nat Protoc 18, 108–135 (2023). https://doi.org/10.1038/s41596-022-00751-1

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